Gene encoding labyrinthin, a marker for cancer

ABSTRACT

A cDNA molecule that encodes a protein designated Labyrinthin (Lab) isolated and its nucleotide sequence is determined. The protein, or peptides derived from the protein, are markers useful to define novel classes of cancers. Diagnostic assays for these cancers use antibodies to Lab or nucleotide probes that hybridize with the lab gene or a fragment therefrom. Vaccines useful either to prevent recurrence of cancers in subjects who test positive for Lab (or lab), or to prevent initial occurrence of cancer, use proteins or peptides derived from Lab. Expression of Lab via immunogenic assays is used to monitor effects of cancer treatments. Antisense molecules against lab are used in treatments. Sense molecules of lab are used to restore lost lab function in diseased normal cells, for example, gland cells.

CROSS-REFERENCE

This application is a continuation application of U.S. Ser. No.12/029,113, filed on Feb. 11, 2008, now U.S. Pat. No. 7,635,759, issuedDec. 22, 2009, which is a continuation of U.S. Ser. No. 10/830,702,filed Apr. 23, 2004, now U.S. Pat. No. 7,329,743, issued Feb. 12, 2008,which is a divisional application of U.S. Ser. No. 09/659,521, filedSep. 12, 2000, now U.S. Pat. No. 6,727,080, issued Apr. 27, 2004, whichis a continuation of PCT/US99/05365, filed Mar. 11, 1999, which is acontinuation-in-part of U.S. Ser. No. 09/040,485 filed Mar. 17, 1998,now U.S. Pat. No. 6,166,176, issued Dec. 26, 2000.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Dec. 8, 2009, is named38964701.txt, and is 10,436 bytes in size.

The invention relates to a gene encoding a protein and peptidestherefrom that includes an epitope, a cancer associated antigen, usefulas a marker that is not restricted to previously defined histologicalclasses of cancer. Antigenic peptides are useful as a vaccine fortreatment and prevention of cancer. Antigenic peptides are useful as avaccine for treatment and prevention of cancer, and for the preparationof new, specific, monoclonal antibodies. Antisense molecules are usefulin pharmaceutical compositions and are useful for diagnosis andtreatment.

BACKGROUND OF THE INVENTION

Cancer¹ is a leading cause of death in men and women throughout theworld. In the United States alone, over 1 million new cases arediagnosed each year, and over 0.5 million deaths are reported annually(Landis, et al., 1998). Historically, tumors are grouped and treated,based in part by the tissues in which they arise, e.g.: breast cancer,colon cancer, and lung cancer, and the like. Yet, within lung cancer,for example, it is well recognized that these tumors are a veryheterogeneous group of neoplasms. This is also true for tumors arisingin other tissues. In part, because of this heterogeneity, there arecomplex and inconsistent classification schemes which are used for humantumors. Previous attempts to treat cancer have been hampered by: 1) thearbitrary classification of tumors arising within given tissues, and 2)by using microscopic methods based on how these tumors look(histological classification). Although existing classifications forvarious tumor types have some prognostic value, almost all of theclassifications fail to predict responsiveness to treatments andlikelihood of cure or disease course. Improved classification schemesbased on the biological constitution of these neoplasms is required tosignificantly alter the survival statistics of humans who have cancer.One approach to solving these problems is to locate molecules specificto tumors, preferably antigens in molecules that are markers for cancercells. (A “marker” is defined herein as any property which can be usedto distinguish cancer from normal tissues and from other diseasestates.) The markers' presence is then a basis for classification.¹Terminology used herein is as follows: “cancer” is a malignant tumor,wherein a “tumor” is an abnormal mass of tissue, that need not bemalignant. “Neoplasm” is a form of new growth.

Monoclonal antibodies (MCAs) prepared by somatic cell hybridizationtechniques, usually in mice, are useful molecular probes for thedetection and discrimination of cellular antigens, and therefore havegreat potential for detecting cancer associated antigens. Theseantibodies bind to specific antigens and the binding is detectable bywell known methods. When binding occurs, the inference is made that aspecific antigen is present. Those cancer associated antigens which areexposed to the cell surface or found in the cancer mass, are moleculartargets for the immune systems (including host antibodies) of the host.Recent findings suggest that cancer patients who have antibodies againsttheir tumors, do better than those who do not mount this type of immuneresponse (Livingston, et al., 1994). Therefore, natural, induced, oradministered antibodies are a promising therapeutic approach.

The humanization of non-human MCAs (the process by which non-human MCAreactive sites are shuttled into cloned human antibodies and expressed)results in reduced immunogenicity of the foreign antibodies without theloss of their specific binding in in vivo and in ex vivo applications.MCAs can be used as in vivo imaging agents, diagnostic tests, and fortherapy (Radosevich, et al. 1988, 1990; Rosen, et al. 1988).

Vaccine therapy is a well established approach directed at inducing animmune response without exposure to the causative agent of a disease orcondition. Many vaccines are available, for example, to stimulate aresponse in a host to bacterial and viral agents. The use of tumorassociated antigens (markers) in a vaccine could prevent primary canceroccurrence, and could also provide a means to prevent recurrence of thedisease.

Gene therapy is a means by which the genetic make-up of cells ismodified to express the gene of interest. There are many forms of genetherapy including: gene replacement, antisense suppression therapy, andsurrogate gene expression. Discovering genes encoding cancer-associated,preferably cancer-specific antigens (markers) opens the door to geneticintervention against cancer cell proliferation. The accurate andconsistent use of a cancer marker to differentiate cancerous from normaltissue, not only has diagnostic potential, but is also desirable fortreatment and prognosis. Therefore, such markers have been sought.

Recent studies have shown that the enzyme encoding human aspartylbeta-hydroxylase (HAAH) is overexpressed in some human adenocarcinomacell lines, and in primary hepatocellular cancers, therefore could be amarker. The gene said to encode HAAH has been cloned and sequenced(Gronke, et al., 1989, 1990; Wang, et al., 1991; Jia, et al., 1992,1994; Korioth, et al., 1994; Lavaissiere, et al., 1996). However, littleis known about HAAH expression in human tumors in general (Lavaissiere,et al., 1996).

The study of the HAAH enzyme grew out of the study of its bovinecounterpart (Gronke, et al., 1989, 1990; Wang, et al., 1991; Jia, etal., 1992). Bovine aspartyl beta-hydroxylase is an intracellular,glycosylated protein, localized in the rough endoplasmic reticulum. Theprotein has been reported to have three major species of molecules; a 85kilodalton form, and two active forms with molecular weights of 56 and52 kilodaltons respectively (Lavaissiere, et al., 1996).

Using standard biochemical methods, bovine aspartyl beta-hydroxylase(bAAH) has been purified and characterized (Gronke, et al. 1990; Wang,et al., 1991). The activity of the enzyme has been shown to becorrelated with the 52 and 56 kilodalton species which were purifiedImmunologically, a related higher molecular weight form (85-90kilodalton) was also observed. As part of the purification, bAAH isbound to Con A sepharose, which is consistent with the conclusion thatthe enzyme is glycosylated. (Subsequent reports on the DNA sequence showthree possible glycosylation sites, with one site being very close tothe known active enzyme domain.) The protein is very acidic in nature,and a detergent is not required to solubilize the active fraction. Theactive enzyme site is dependent from the biochemically isolated bovineprotein (bAAH) on the presence of histidine at position 675 (Jia, etal., 1994).

A partial amino acid sequence was obtained for HAAH. DNA probes (a DNAprobe is a molecule having a nucleotide sequence that is capable ofbinding to a specified nucleotide sequence) deduced from this amino acidsequence was used to screen a bovine cDNA library (Jia, et al., 1992).(A cDNA library contains the sections of DNA that encode for geneproducts, e.g. peptides, as opposed to genomic DNA). Several overlappingcDNA sequences in the library contained a 754 amino acid open readingframe (ORF) sequence which would be expected to encode an 85 kilodaltonprotein. Also present in this ORF sequence were two other possible startcodons, that is, locations at which encoding begins. The most 3′ startcodon was preceded by a ribosome binding site. Translation of the clonehaving this sequence resulted in a protein that was about 85kilodaltons. Antiserum was raised to the membrane fraction of humanMG-63 cells and was used to immunoscreen a cDNA library made from MG-63cells. Data on one clone was reported which could encode a 757 aminoacid protein, and, by sequence analysis, was found to have strongN-terminal homology with bAAH (Korioth, et al., 1994). When this clonewas used in an in vitro translation system (an artificial cocktail ofnormal cell cytoplasm used to convert mRNA into protein), a 56kilodalton protein was produced. It was suggested that this was due toposttranslational cleavage.

The HAAH enzyme is responsible for the modification of specific asparticacid residues within the epidermal growth factor-like domains ofproteins. It has been hypothesized that these modified aspartic acidresidues allow the epidermal growth factor-like domains to becomecalcium binding domains (Gronke, et al., 1989, 1990; Wang, et al., 1991;Jia, et at, 1992, 1994; Korioth, et al., 1994; Lavaissiere, et al.,1996).

An enzyme related to HAAH, aspartyl beta-hydroxylase (AAH), was firststudied because it specifically modified select aspartic acid orasparagine residues in a group of biologically important proteinsincluding the vitamin K-dependent coagulation factors VII, IX, and X.Other proteins like C, S, and Z also have this modification (Gronke, etal., 1989, 1990; Wang, et at, 1991; Jia, et al., 1992, 1994; Korioth, etal., 1994; Lavaissiere, et al., 1996). Aspartic acid and asparagineresidues have been shown to be modified by HAAH in proteins containingepidermal growth factor-like domains. The function of thebeta-hydroxyaspartic and beta-hydroxyasparagine residues is unknown,however, it has been speculated that this modification is required forcalcium binding in the epidermal growth factor EGF-like domains ofselected proteins.

Antibodies were raised to human hepatocellular carcinoma FOCUS cells(Lavaissiere, et at, 1990). One MCA reacted with an antigen that washighly expressed in hepatocellular carcinomas (Lavaissiere, et al.,1996). Immunoscreening using this antibody and a lambda gt11 HepG2library resulted in the isolation of a partial cDNA, which wassubsequently used to isolate a larger clone.

A human adenocarcinoma cell line designated A549 was reported as havingvery high levels of HAAH activity (Lavaissiere, et al., 1996). A mousemonoclonal antibody designated 1.1 MCA 44-3A6 (U.S. Pat. No. 4,816,402)was produced against the human adenocarcinoma cell line A549 (ATCCaccession number CCL 185) (Radosevich, et al., 1985). The antibodyrecognized a cell surface, non-glycosylated antigenic protein having anestimated apparent molecular weight of 40 kDa).

The antigen was expressed by A549 cells, and was found to be a goodadenocarcinoma marker; that is, it was frequently expressed by cancerswhich looked like adenocarcinomas when examined histologically(Radosevich, et al., 1990a; Lee, et al., 1985). MCA 44-3A6 is unique inthat it is the first monoclonal antibody which has this bindingspecificity. The results from an International Workshop for Lung cancerconfirmed other related published findings on MCA 44-3A6 (Stahel, 1994).

The antibody designated MCA 44-3A6 has clinical utility because itdifferentiates antigens associated with adenocarcinomas. The normal andfetal tissue distribution of the antigen is restricted to some glandulartissues (Radosevich, et al., 1991). Detection can occur on formalinfixed-paraffin embedded tissue (Radosevich, et al., 1985, 1988, 1990a,1990b; Lee, et al., 1985, 1986; Piehl, et al. 1988; Combs, et al.,1988b, 1988c; Banner, et al., 1985). The antibody has a restrictedbinding pattern within human pulmonary tumors (Lee, et al., 1985;Banner, et al., 1985; Radosevich, et al., 1990a, 1990b).

In a study of over two hundred pulmonary cancers, MCA 44-3A6 was foundto react with all of the adenocarcinomas tested, many of the large cellcarcinomas, as well as with subsets of intermediate neuroendocrine smallcell lung cancers, well-differentiated neuroendocrine small cellcarcinomas, carcinoids, but not mesotheliomas. MCA 44-3A6 does not reactwith squamous cell carcinoma, bronchioloalveolar carcinoma, or smallcell carcinoma (Lee, et al., 1985). MCA 44-3A6 is useful indistinguishing adenocarcinomas that are metastatic to the pleura frommesothelioma (Lee, et al., 1986). The antibody has selected reactivityamong adenocarcinomas and in large cell carcinomas (Piehl, et al., 1988;Radosevich, et al., 1990b).

In a study of over 40 cases of lung cancer comparing cytological andhistological findings, MCA 44-3A6 was found to be useful in cytologicaldiagnosis and was consistent with the histological finding (Banner, etal., 1985). Histology is the study of tissues (which are made of cells).Cytology is the study of cells which have been removed from theorganizational context which is commonly referred to as tissue. Cellsremoved from tissues do not always behave the same as if they were inthe tissue from which they were derived. Fortunately, the antigendetected by MCA 44-3A6 expressed in adenocarcinoma cells in tissuebehaves in the same ways as adenocarcinoma cells removed from tissues.This is a very diagnostically important characteristic. Similarcorrelations using cytologically prepared cell blocks of pulmonarycarcinomas, as well as ACs presenting in body fluids from other sitesthroughout the body were demonstrated (Lee, et al., 1985; Spagnolo, etal., 1991; Combs, et al., 1988c). Also, MCA 44-3A6 binds toadenocarcinomas from sites other than lung cancer. The expression of theantigen in primary and metastatic lesions was also reported (Combs, etal., 1988a). The utility of the MCA antibody in differentiating cancerfrom benign lesions in human breast tissue was also noted (Duda, et al.,1991).

The cellular localization of the antigen detected by MCA 44-3A6 wasdetermined. By using live cell radioimmunoassays (a radioactive antibodytest directed at determining binding of the antibody to live cells),immunofluorescence, and live cell fluorescence activated cell sorter(FAGS) analysis, the antigen detected by MCA 44-3A6, was shown to be onthe outside surface of the cell (Radosevich, et al., 1985). Additionalstudies using immunogold-electron microscopy and FACS analysis havedemonstrated that this antigen is non-modulated (that is notinternalized by the cancer cell when bound by an antibody), is expressedon the extracellular surface of the plasma membrane, and is not cellcycle specific that is, the cell makes protein all the time it is goingthrough the process of cell replication, and also when it is notdividing (Radosevich, et al., 1991). The antigen is not found in theserum of normal or tumor bearing patients, and is not shed into theculture media by positive cell lines (that is, cancer cells are known tobleb off portions of their cell membranes and release them into thesurrounding fluid.) (Radosevich, et al., 1985). Recently 3 of 27randomly tested adenocarcinoma patients were found to have naturallyoccurring antibodies to the antigen. In addition, radiolabeled MCA44-3A6 was used to localize A549 tumors growing in nude mice. Adouxorubicin immunoconjugate MCA 44-3A6 is selectively toxic in vitro(Sinkule, et al., 1991).

Determination of the nucleotide and amino acid sequences of the antigendetected by MCA 44-3A6 would enhance the usefulness of this antigen incancer diagnosis, treatment and prevention

SUMMARY OF THE INVENTION

The antigen detected by the antibody MCA 44-3A6 as described in theBackground is now designated as “Labyrinthin.” A gene (designatedlabyrinthin; abbreviated lab) characterized by a unique nucleotidesequence that encodes the antigen detected by MCA 44-3A6 was isolatedand characterized. (lab notation signifies the nucleic DNA/RNA forms;“Lab” notation refers to the protein which is encoded by the labDNA/RNA).

The invention described herein used the antibody MCA 44-3A6 as a tool toclone the gene encoding Lab. In addition, an epitope (the necessarybinding site for an antibody found on the antigen) for MCA 44-3A6 wasidentified on the Lab protein expressed by the clone to be PTGEPQ² (SEQID NO: 10). The epitope represents an important immunodominant sequence;that is, when injected into animals, the animals readily produceantibodies to this sequence. ²Standard abbreviations for amino acids.

An aspect of the invention is the use of lab DNA in the sense³expression mode for: 1) the marking of human tumors by nucleotideprobes; 2) the detection of DNA and mRNA expression of lab in cells andtissues; 3) the transformation of cells into a glandular-like cell type;4) the production of Lab antigen in vivo for immunization; 5) the exvivo expression of Lab for immunization to produce antibodies; and 6)production of Lab in vitro. Use of an antisense molecule, e.g. byproduction of a mRNA or DNA strand in the reverse orientation to a sensemolecule, to suppress the growth of labyrinthin-expressing (cancerous)cells is another aspect of the invention. ³The normal transcription of aDNA sequence which proceeds from the 3′ to the 5′ end to produce a mRNAstrand from the sense strand of DNA, the mRNA being complementary to theDNA.

An aspect of the invention is a vector comprising a DNA molecule with anucleotide sequence encoding at least an epitope of the Lab antigen, andsuitable regulatory sequence to allow expression in a host cell.

Another aspect of the invention is an amino acid sequence deduced fromthe protein coding region of the lab gene. Select regions of thesequence were found via immunological methods, to correspond and reactto both naturally occurring (from cancer cells), chemically produced(synthetically produced peptides), and the expression of the cloned labgene.

Another aspect of the invention is the use of the entire deduced aminoacid sequence of Lab, peptides derived from Lab, or chemically produced(synthetic) Lab peptides, or any combination of these molecules, for usein the preparation of vaccines to prevent human cancers and/or to treathumans with cancer. For purposes of the present invention, “humans withcancer” are those persons who have the Lab antigen detected on theircells. These preparations may also be used to prevent patients from everhaving these tumors prior to their first occurrence.

Monoclonal antibodies directed to the Lab protein, or antigen componentsor derivatives of Lab proteins, are useful for detection of Lab and forother purposes. Monoclonal antibodies which are made in species otherthan those which react with the Lab antigen can be modified by a numberof molecular cloning such that they retain their binding with theLabyrinthin peptides, yet are not immunogenic in humans (Sastry, et al.,1989; Sambrook, et al., 1990). In brief, this is done by replacing thebinding site sequence of a cloned human antibody gene, with the bindingsite sequence of the non-human monoclonal antibody of interest. These“humanized” MCAs are used as therapeutic and diagnostic reagents, invivo, ex vivo, and in vitro.

The use of the Lab protein or antigenic peptides derived therefrom indiagnostic assays for cancer is a way to monitor patients for thepresence and amount of antibody that they have in their blood or otherbody fluids or tissue. This detection is not limited to cancers of aclass or classes previously defined, but is useful for cancer cells thathave the Lab marker antigen. The degree of seroconversion, as measuredby techniques known to those of skill in the art [e.g. ELISA (Engralland Perlmann, 1971)] may be used to monitor treatment effects.

Treatment with antisense molecules to lab or antibodies to Lab is anapproach to treat patients who have Lab in, or on, their cancer cells.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 is the nucleic acid sequence (SEQ ID NO: 1) of the lab gene.

FIG. 2 is the amino acid sequence (SEQ ID NO: 2) for Lab, deduced fromthe lab gene. FIG. 2 also discloses residues 25-52 of SEQ ID NO: 2 andSEQ ID NOS 3-5, respectively, in order of appearance.

FIG. 3 is an illustration of the lab gene and how it is related to theHAAH enzyme.

DETAILED DESCRIPTION OF THE INVENTION

Molecular Biology of Labyrinthin: To demonstrate that the MCA 44-3A6epitope is encoded by a protein sequence, high molecular weight DNA fromthe cell line A549 was isolated. This DNA was co-precipitated (viacalcium) with a plasmid (pSVneo), and used to transfect a mouse cellline designated B78H1 cells (Albino, et al., 1985). This mouse cell lineis negative for the expression of the epitope and was reported to have ahigh frequency of incorporation and expression of any human DNAsequences. If a given B78H1 cell was in a state to take up DNA, it wouldbe expected to have taken up both human DNA and the plasmid DNA. Theplasmid DNA makes the cell resistant to G418 (a normally toxic drug).Therefore, if a cell normally sensitive to G418 growth inhibitor growsin G418, it had to have taken up the plasmid, and may also have taken upone or more A549 DNA sequences. After G418 selection (a way of choosingonly cells which have resistance to growth in G418 by theuptake/expression of the Neo gene on pSVneo plasmid, and thereforerepresenting cells that were in a state to uptake other DNA at the sametime), approximately 15 of 1×105 clones were detected usingimmunoselection with MCA 44-3A6. This finding is consistent with aconclusion that human A549 cells have DNA that encodes Lab and possessesthe regulator sequences necessary for the expression of Lab.

Comparison of HAAH and Labyrinthin: Because the DNA sequence of lab wasdetermined as an aspect of the present invention, HAAH and lab could becompared. HAAH and the lab nucleotide sequences have some internalfragment similarities, but are different on either side of the fragment,and are related to different products. This conclusion is based in partby the—analysis and homology of the DNA sequences reported for these twogenes. Specifically, the lab 5′ region has no homology with HAAH. Theprotein coding region of lab has about a 99.6% homology with an internalsegment of the proposed protein coding region for HAAH. The 3′ regionhas no homology with the HAAH reported sequence. Virtually all of theother data comparing HAAH and labyrinthin are different, for example: 1)molecular weights of the proteins, 2) cellular localization, 3)chromosome localization, 4) histological presentation in normal tissuesand tumors, 5) northern blot expression, 6) immunological findings.

Although the protein coding region of lab is identical to an internalregion of the sequence reported for HAAH, the 5′ untranslated region ofHAAH is different; and part of the 5′ translated protein coding regionof HAAH is missing from that found in the lab clone. From both HAAH andlab clones, the deduced protein would be expected to be very acidic innature, and therefore would run anomolously in SDS gels. As predicted,the Lab protein migrates anomolously in SDS gels. What was cloned anddisclosed in the present invention migrates identically to the nativeprotein found in several cell lines. Convincing evidence that thecorrect gene fragment encoding the antigen detected by MCA 44-3A6 hasbeen cloned (mRNA) is that when the recombinant protein is made, thatrecombinant protein should act (in this case—have an apparent molecularweight) the same as independent biologically derived source of thatprotein. Lab provided from clones has the characteristics of Lab fromcells.

The deduced amino acid sequence encoded by HAAH requires the use of anopen reading frame which would produce a protein that is 85-90kilodaltons, and does not take into account that there are several startcodons and other shorter open reading frames. The deduced HAAH protein(biochemically) is glycosylated and the reported sequence hasglycosylation sites (Korioth, et al., 1994; Lavaissiere, et al., 1996).To the contrary, Lab is not glycosylated, nor does it have predictedglycosylated sites.

The deduced HAAH amino acid sequence contains a region shared by the Labamino acid sequence which is predicted to be very hydrophobic. Labrequires strong detergents in order to be soluble; HAAH does not. Theincreased expression of HAAH (by enzyme activity measurements) in thesame cell line (A549) which was used to clone and study lab extensively,suggests that both of these gene products may be important to the ACphenotype and that at least A549 cells make both functional HAAH andLab. Successful transfections of the antisense to lab into A549 resultedin a marked decrease in expression of lab and in the growth rate of thecells. The expression of a sense lab construct in NIH-3T3 cells (normalmouse fibroblasts) resulted in a marked change in phenotype, a phenotypeconsistent with that of ACs. Therefore, lab expression is associatedwith conversion of normal cells to cancerous cells. Lab and HAAH havepotential calcium binding domains in common.

cDNA Library Construction and Cloning: A cDNA lambda gt11 phage librarywas constructed using mRNA which was isolated from actively growing A549cells (Sambrook, et al., 1990). This oligo(dT)-primed cDNA was clonedinto the Eco RI site using Eco RI linkers. The library has about 83%clear (containing an insert) plaques with a titer of 1.2×10¹⁰/mlrepresenting a minimum of 1.46×10⁶ independent plaques which, byPolymerase Chain Reaction, have insert sizes ranging from 0.6 to 5kilobases. Since Lab is a 40 kilodalton integral protein, (a proteinwhich is embedded in the plasma membrane,) the theoretical full lengthmRNA encoding this protein, including a potential leader sequence isestimated would be about 1.1 kilobases. This library was immunoscreenedusing the antibody MCA 44-3A6. Eight independently derived phase stocks(identical phage which are from the same plaque) were isolated. Thesehave all been plaque purified by repeated cycles ofimmunoscreening/isolation. Upon Eco RI digestion of these eightisolates, inserts of about 2 kb were seen. The largest insert wasisolated (2A1 A1) and the Eco RI fragment was cloned into the pGEM-3Zplasmid.

Sequencing and Sequence Analysis: The DNA fragment designated 2ALA wasfound to have an insert of 2442 base pairs in length (FIG. 1),containing a 5′ untranslated region, a ribosome binding site, and astart codon which would be expected to encode a 255 amino acid protein(FIG. 2). The 3′ untranslated region is remarkable in that it containsonly four instability sequences; ATTTA (Xu, et al. 1997). In additionthere are sequences found in the very 3′ end of mRNA's which result inadenylation of the mRNA (Sambrook, et al., 1990). The lab sequencecontains both a sub-optimal (ATTAAA) and optimal (AATAAA)poly-adenylation site. These are sequences found in the very 3′ end ofmRNA's which result in adenylation of the mRNA. This finding providesmolecular data which supports the cellular and biochemical data that hasbeen outlined herein. (The HAAH clone has a poly A signal, but the whole3′ region has not been sequenced.)

A calcium binding site motif is noted in the Lab amino and sequence(FIG. 2), however, it is out of the known required structural context tobe a binding site. In this case, the calcium limiting sequence is there,but it is not in a protein sequence context that is known to make itwork as a binding site! Homology was noted with lab and an EST clone(designated #05501) which represented only a portion of the 3′untranslated region and independently confirmed this portion of thesequence. Some internal fragment homology is also noted with HAAH, butthe 5′ untranslated and part of the 5′ translated region is different(58 amino acids), as well as a major portion of the 3′ coding region ismissing in lab (FIG. 3).

Genomic DNA Cloning and Analysis: Using a PCR fragment representing theprotein coding region of lab as a probe, a genomic lambda FIX II librarymade from the human pulmonary fibroblast cell line WI-38 was screened.Ten primary plaques were isolated out of approximately 1×10⁶ screenedplaques. Using seven of these as target DNA, Polymerase Chain Reactionconditions were established with primers for the protein coding region,producing a 765 base pair fragment, the expected protein coding regionfor lab. On Northern blots (a method used to qualitatively assess mRNA)lab only detects one band noted at 2.7 kilobases. The recombinantprotein made from the lab clone, when tested on Western blots (a methodused to qualitatively define proteins) using MCA 44-3A6, has the samerelative mobility as the Lab protein when made by A549 cells.

Lab and HAAH genes give different results in the proteins they encode.HAAH consistently gives two bands on Northern blot analysis (2.6 and 4.3kilobases) suggesting that the 2.6 kilobase band is due to alternativesplicing, i.e. the cell cuts and splices the mRNA. Also, if lab and HAAHare the same gene, HAAH should be detected in all tissues and cancercell lines in which Lab is found. However, Lab is not seen on Northernblots of cell lines EMT6 or QU-DB, nor is there immunoreactivity inthese cells; indicating that Lab mRNA is not made, and that Lab proteinis not produced in these cells. Lab protein is rarely expressed innormal cells, where both the HAAH mRNA and HAAH protein have beenreported to be expressed in almost every tissue studied.

mRNA Analysis: Northern blot analysis of the DNA fragment from the A549cell line using lab cDNA as a probe identified a single band of about2.7 kilobases. This is expected based on the cDNA (2442 base pairs) anda poly-A tail of about 300 base pairs. Northern blot analysis of themouse cell line, EMT6, and of the human large cell carcinoma cell line,QU-DB, confirm that no transcript for lab is produced by these cells.This is consistent with immunoassays which are negative for labexpression on these cells.

Antisense and Sense cDNA Expression: The plasmid (pBK-CMV) (Sambrook, etal., 1990) may carry either the sense or antisense full length cDNA labinto A549 and NTH 3T3 cells. An antisense molecule can be, for example,a complementary sequence to a sense molecule that hybridizes with thesense molecule, preventing its expression. Using the MIT assay(Siddique, et al., 1992) to assess the growth rate of A549 cellsexpressing antisense to lab, a marked reduction in growth rate wasnoted. The antisense transfected A549 cells appear to have a greaterdegree of contact inhibition. A detectable amount of Lab is reduced inthese antisense transfected cells. NIH-3T3 cells convert from afibroblast-like cell type morphology (large, thin spindle shaped) to alarge, adenocarcinoma appearing cells (very round, plump) when senseexpression occurs.

Chromosome Localization: The chromosome localization for lab, using fulllength cDNA as a probe via in situ hybridization (Sambrook, et al. 1990)is tentatively on chromosome 2q12-14, with possibly some reactivity tochromosomes 4 and 8. Using the same probe (the full length cDNA sequenceof lab) and FACS sorted chromosomes (Lebo, et al. 1985) staining wasalso noted on chromosome 2, with weak staining on 4 and none on 8. Theuse of genomic clones will be of particular value in resolving thesedata because higher stringency hybridization conditions than thatallowable for the cDNA, can be used, thereby reducing backgroundsignals. This is yet another proof that the correct gene was cloned andthat the results are not due to a method artifact. There may bemutations in the genomic DNA of tumors and for the present invention,DNA was cloned from tumor cells (A549). Therefore, a mutated gene couldhave been cloned. However, that is not the case because the genomic DNAfrom a normal cell (DNA) produced the same sequence as what cloned asdescribed herein. Therefore, a normal gene was cloned from A549 cells.The weak signals on chromosomes 4 and 8 are consistent with a pseudogeneor a related gene. For example, HAAH has been reported to be onchromosome 8q12 by in situ hybridization, so this result on chromosome 8could reflect the HAAH and lab sequence homology.

Protein Molecular Characterization of Labyrinthin: Previous work usingWestern blot analysis (a qualitative assay to assess antigens) has shownthat the Lab antigen is a 40 kilodalton (by relative mobility) proteindetectable in A549 cells (Radosevich, et al., 1985). The epitope doesnot appear to be modulated or blocked by lectins, and is selectivelyexpressed on the cell surface, primarily localized to the plasmamembrane. (Radosevich, et al., 1985, 1991). Lab is sensitive toproteases, but not lipid or carbohydrate altering reactions (Radosevich,et al., 1985). The biochemical properties of Lab are consistent with Labbeing an integral membrane protein.

Having a deduced amino acid sequence from the lab gene of the presentinvention, allows further characterization of the Lab protein. Extensivecomputer analysis of Lab has identified a eukaryotic leader-likesequence and theoretical cleavage site, 3 myristylation sequence sites,a weak membrane anchoring domain (MAD I), and a strong membraneanchoring domain (MAD II) (FIG. 2). [(In the HAAH sequence, there are 58(theoretical) amino acids followed by a sequence homology in the Labprotein coding sequence, and an additional 445 amino acid 3′ to the labsequence.)]

When Lab is expressed as a fusion protein in a bacterial GST fusionexpression system (pGEMEX-2T) (Amereham Pharmacia Biotech, Inc.,Piscataway, N.J., 08854, USA), and subjected to Western blot analysisusing the antibody MCA 44-3A6, the resulting blots demonstrate that theexpressed cleaved fusion protein has the same relative mobility as theprotein detected in A549 cells. The deduced molecular weight for Lab is28.8 kilodaltons and on Western blots it has a relative mobilityidentical to the form expressed by A549 cells (apparent relativemobility=40 kilodaltons). The 55 glutamic and 27 aspartic acid residues(82 residues combined) are almost uniformly distributed throughout theprotein (255 amino acids total; 228 no leader sequence), except for theleader sequence and the strongest membrane anchoring domain (MAD II).These data suggest that Lab migrates anomalously in SDS gels. Cell linesother than A549 (e.g. adenocarcinomas DU-145, ATCC #HTB-81; ZR-75-1,ATCC #CRL-1504, and so forth) have an antigen detected with the samemolecular weight antigen as Lab. Neither a 85-90 kilodalton molecularweight species, nor a 52 and 56 kilodalton molecular weight species isnoted when probing Western blots for Lab.

Epitope Mapping Using the Antibody MCA 44-3A6 and Vaccine Feasibility ofLab: Using Polymerase Chain Reaction and the GST fusion protein system,subclones of the protein coding region were made, and epitopes mappedthe binding of MCA 44-3A6 to six amino acids (PTGEPQ) (SEQ ID NO: 10)representing amino acids #117-122 of Lab (“P” peptide). In order todetermine this epitope, the entire coding region was divided intoregions, Polymerase Chain Reaction primers were designed to amplify eachregion, and the subsequent expression of Polymerase Chain Reactionproducts were cloned and tested by Western blot analysis using theantibody MCA 44-3A6.

The DNA fragment representing the positive Western blot result was thenfurther subdivided. Polymerase Chain Reaction products were generatedand cloned, expressed, and tested via Western blot. Constructs were madein this way both from the 5′ end and the 3′ end and the intervals of thenumber of amino acids were reduced upon each round. This resulted in thelast round representing a one amino acid difference from the previousround (in both directions), such that one could deduce the exact bindingsite of the MCA 44-3A6. This demonstrates that at least these six aminoacids are exposed to the external cell surface. To further prove thepoint, the DNA encoding only these six amino acids have been cloned andthe fusion protein is positive by Western blot analysis. Syntheticallyprepared “P” peptide can be specifically detected by MCA 44-3A6, and thesynthetic peptide was immunogenic in 5 of 5 mice tested. Computeranalysis/modelling also predicted that this epitope would be veryimmunogenic using computer assisted analysis (GCG programs) (GeneticsComputer Group, Madison, Wis. 53703).

Vaccine Preparation: A vaccine is a preparation of antigen(s), whichwhen given to a host, results in the host producing antibodies againstthe antigen(s). The host response results in the host being immune tothe disease to which the vaccine was directed. Vaccine treatmenttherefore, prevents the clinical presentation of a disease, without thehost being exposed to the disease causing agents. Lab has all thecharacteristics of a preferred cancer vaccine. The lab gene isfrequently expressed by tumors which look like adenocarcinomas, isexpressed on the outside of the cells, is expressed by all of the cellswithin a given cancer, is expressed at all times by these cancer cells,and is infrequently expressed by normal cells. Lab protein (peptides)can be produced by any number of methods using molecular cloningtechniques, and can be produced in large quantities, thus making it apractical antigen to use as a vaccine. After the Lab protein has beenpurified so that it is suitable for injection into humans, it isadministered to individuals intradermally, subcutaneously, or by otherroutes, so as to challenge the immune system to produce antibodiesagainst this protein (peptides).

The use of molecular modeling and computer assisted analysis GCGprograms (Genetics Crystal Group, Madison, Wis. 53703) allows theidentification of small portions of a molecule, slightly larger than anepitope (six to seven amino acids for proteins), which are expected tobe on the surface of a protein molecule. In addition, the degree ofhydrophobicity or hydrophilicity of a given sequence, and howimmunogenic the sequence would be in animals, can be determined(Genetics Crystal Group, Madison, Wis. 53703). After defining whichsequences meet these criteria, the peptides are synthetically made, orproduced by a number of standard methods. One or more of these peptidescan then be formulated to be used as a vaccine, and administered to thehost as outlined above, as a vaccine.

A vaccine comprising a molecule having an amino acid sequence selectedfrom the group of sequences encoded by the cDNA of FIG. 1, sequences ofFIG. 2, encoded by the cDNA, the peptides APPEDNPVED (SEQ ID NO: 6),EEQQEVPPDT (SEQ ID NO: 7), DGPTGEPQQE (SEQ ID NO: 8) and EQENPDSSEPV(SEQ ID NO: 9), and any fragments, or combinations thereof.

A given vaccine may be administered once to a host, or may beadministered many times. In order for some patients to recognize a givenvaccine, an adjuvant may also need to be administered with the peptides.Adjuvants are nonspecific immune stimulators which heighten the immunereadiness and aid in the conversion of the host from not havingdetectable serum antibodies to having very high titer serum antibodies.It is this high level (titer) of antibodies, which effectively protectsthe host from the diseases or conditions to which the antibodies aredirected.

Functional Studies: Studies directed at understanding the cellularfunction(s) of Lab are extensions of cell localization/characterizationstudies (Siddique, et al. 1992). Changes in levels of Lab in cations(Ca++, Mg++, Cu++, and Fe++) were undertaken. Lab expression in A549cells was only modulated by Ca++. Using the highly specific fluorescentFura-2/AM Ca++ method of measuring cytosolic Ca++, (Molecular ProbesInc., Eugene, Oreg. 97402) it was demonstrated that: 1) the internalCa++ concentration is higher in A549 cells than in QU-DB cells, and 2)that the A549 cell line responds to various external Ca++ levels(Siddique, et al., 1992). Since pH can modulate intracellular free Ca++levels, external pH manipulations should result in changes in theexpression levels of Lab. Extracellular pH changes (in the presence ofnormal Ca++ concentrations) result in 1) a parallel change inintracellular pH as measured by SNARF-1 AM/FACS, (Molecular Probes Inc.,Eugene, Oreg. 97402) 2) transcript levels increase for Lab (whencompared to GAPDH expression via Northern blot), and that 3) Lab proteinalso increases (using Western/Slot blot analysis). The intracellularchanges in pH (due to external changes) for A549 cells are identical tothose reported for normal cells. The increased expression of lab is alsonot due to cell death (as measured by MTT assays) (Siddique, et al.,1992). In addition, incubation of recombinant Lab at various pHsolutions does not alter immunoreactivity. Preliminary data suggeststhat when these experiments are conducted on A549 cells grown in reducedCa++, the induced expression of lab is blunted.

Methods of Diagnosing Cancer Cells in a Sample of Cells: Biologicalsamples from a subject are used to determine whether cancer cells arepresent in the subject. Examples of suitable samples include blood andbiopsy material. One method of diagnosis is to expose DNA from cells inthe sample to a labeled probe that is capable of hybridizing to the labgene, or a fragment thereof, under stringent conditions, e.g. 6× ssc;0.05× blotto; 50% formamide; 42° C. (Sambrook, et al., 1990). Of course,the hybridizing conditions are altered to achieve optimum sensitivityand specificity depending on the nature of the biological sample, typeof cancer, method of probe preparation, and method of tissuepreparation.

After contacting the sample with the probe, the next step is determiningwhether the probe has hybridized with nucleotide sequences of the DNAfrom the sample, from which the presence of the lab gene is inferred,said presence being diagnostic of cancer.

Another diagnostic method is to obtain monoclonal antibodies preferablylabeled, either antibodies already existing, or new ones directed to theantigenic peptides that are aspects of the present invention, andcontact a sample with these to detect the Lab antigen. These monoclonalantibodies are useful in the development of very specific assays for thedetection of Lab antigen, and allow the tests to be carried out in manydifferent formats; resulting in a broader application in science andmedicine.

The current invention is useful in that it describes a new gene which isexpressed on the surface of tumors, which was not previously reported.This gene is not tissue specific, and therefore will allow the detectionof tumors regardless of the organ in which they arise. Likewise, the useof this gene to produce a vaccine for these tumors, will have a verybroad application. Diagnostic tests will also have this broad tissueuse, making the detection of Lab//ab a “pan-marker” for cancer, inparticular for what have been designated previously, adenocarcinomas.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

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1. An isolated monoclonal antibody or antigen-binding antibody fragmentthereof directed specifically to an epitope contained in (1) apolypeptide encoded by a cDNA molecule having the nucleotide sequence ofSEQ ID NO: 1, wherein said epitope is not PTGEPQ (SEQ ID NO: 10), or (2)a polypeptide having the amino acid sequence of SEQ ID NO: 2, whereinsaid epitope is not PTGEPQ (SEQ ID NO: 10).
 2. The antibody of claim 1,wherein the antibody is a humanized antibody.
 3. The antibody of claim1, wherein the epitope is predicted to be on the surface of a proteinmolecule by use of GCG molecular modeling and computer assistedanalysis.
 4. The antibody of claim 1, wherein the antibody is humanizedand is directed specifically to a peptide consisting of the amino acidsequence APPEDNPVED (SEQ ID NO: 6).
 5. The antibody of claim 1, whereinthe antibody is humanized and is directed specifically to a peptideconsisting of the amino acid sequence EEQQEVPPDT (SEQ ID NO: 7).
 6. Theantibody of claim 1, wherein the antibody is humanized and is directedspecifically to a peptide consisting of the amino acid sequenceEQENPDSSEPV (SEQ ID NO: 9).
 7. The antibody of claim 1, wherein theepitope is within a predicted calcium binding region consisting of aminoacids 25 to 52 of SEQ ID NO:
 2. 8. A pharmaceutical compositioncomprising an isolated monoclonal antibody or antigen-binding antibodyfragment thereof directed specifically to an epitope contained in (1) apolypeptide encoded by a cDNA molecule having the nucleotide sequence ofSEQ ID NO: 1, wherein said epitope is not PTGEPQ (SEQ ID NO: 10), or (2)a polypeptide having the amino acid sequence of SEQ ID NO: 2, whereinsaid epitope is not PTGEPQ (SEQ ID NO: 10).
 9. The composition of claim8, further comprising an adjuvant.
 10. The composition of claim 8,wherein the antibody is a humanized antibody.
 11. The composition ofclaim 8, wherein the antibody is directed specifically to a peptideconsisting of the amino acid sequence APPEDNPVED (SEQ ID NO: 6).
 12. Thecomposition of claim 8, wherein the antibody is directed specifically toa peptide consisting of the amino acid sequence EEQQEVPPDT (SEQ ID NO:7).
 13. The composition of claim 8, wherein the antibody is directedspecifically to a peptide consisting of the amino acid sequenceEQENPDSSEPV (SEQ ID NO: 9).
 14. A method of forming an immunocomplexcomprising contacting an adenocarcinoma cell with an antibody orantigen-binding antibody fragment thereof directed specifically to anepitope contained in (1) a polypeptide encoded by a cDNA molecule havingthe nucleotide sequence of SEQ ID NO: 1, wherein said epitope is notPTGEPQ (SEQ ID NO: 10), or (2) a polypeptide having the amino acidsequence of SEQ ID NO: 2 to generate an immunocomplex, wherein saidepitope is not PTGEPQ (SEQ ID NO: 10).